Properties of [3H]flunitrazepam binding to different benzodiazepine binding proteins

Membranes from cerebellum or hippocampus were incubated with various concentrations of [3H]flunitrazepam in the absence or presence of diazepam, Cl 218 872 or ethyl-beta-carboline-3-carboxylate (beta-CCE). After binding equilibrium of [3H]flunitrazepam had been established, the membranes were either filtered for determination of reversible binding or were irradiated with UV light for determination of irreversible binding. Irradiated membranes were then subjected to SDS-polyacrylamide gel electrophoresis and fluorography. Individual photolabeled proteins were identified, appropriate sections cut out of the gel, and the radioactivity in the gel pieces measured. The results indicate that [3H]flunitrazepam binding to individual benzodiazepine binding proteins and its inhibition by various drugs can be measured by the present technique and support previous evidence for the independent existence of various proteins irreversibly labeled by [3H]flunitrazepam and their possible association with different benzodiazepine receptors.